Suramin and derivatives thereof as topical microbicide and contraceptive

ABSTRACT

The present invention is directed to methods of inhibiting STDs by topically administering suramin or a derivative thereof to actual or potential sites of infection, and methods of preventing pregnancy by topically applying suramin or a derivative thereof intravaginally. Suramin compositions that include an antimicrobial agent and/or a sperm-function inhibitor are also provided and may advantageously be used in the methods of the invention. A method of simultaneously inhibiting STDs and preventing pregnancy is also provided. Devices impregnated or coated with the topical suramin compositions are further disclosed and may be used to apply the compositions described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/367,273, filed on Mar. 26, 2002, which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded in part by the Contraceptive Research andDevelopment (CONRAD) Program (grant number HRN-A-00-98-00020-00),established as a cooperative agreement between the U.S. Agency forInternational Development (USAID) and the Eastern Virginia MedicalSchool (EVMS). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention generally relates to suramin and its derivatives intopical formulations and their use as microbicidal contraceptives forthe prevention of sexually transmitted diseases (STD) and conception.According to the present invention, it has been found herein thatsuramin and its derivatives display potent activity against STDpathogens and has displayed antisperm activity, and alone or incombination with other sperm-function inhibitors, including spermicides,and/or other antimicrobials, including antivirals, they constituteeffective vaginal contraceptive microbicides.

The increasing prevalence of STD is a serious public health problemaffecting both developed and less-developed countries. In the latter,the acquired immune deficiency syndrome (AIDS) epidemic is taking adevastating toll in human lives. Constrained by lack of money,overpopulation and cultural habits that make condoms unpopular, thesecountries see a dramatic increase in the number of humanimmunodeficiency virus (HIV)-infected people, in some cases reaching onethird of the population of reproductive age. There is an urgent need todevelop safe, prophylactic, female-controlled agents that are effectiveagainst sexually transmitted pathogens, particularly HIV, andconception.

Nonoxynol-9 (N-9), a nonionic surfactant, is the only FDA-approvedspermicide currently in the U.S. market. Other surfactants, such asbenzalkonium chloride, are available as part of spermicidal preparationsin Canada and Europe. Due to their effects on lipids, these surfactantsdisplay anti-HIV activity in vitro, disrupting the viral envelope andinactivating the virus. Unfortunately, as it has clearly beendemonstrated for N-9, these surfactants alone do not appear to confersignificant protection in vivo. Several clinical trials with N-9 haveshown lack of reduction in the incidence of HIV infection (Rowe,Lancet., 349: 1074 1997; Hira et al., Int J. STD AIDS., 8(4) 243-501997; Martin et al., Sex. Transm. Dis., 24(5): 279-283, 1997; Roddy etal., N. Engl. J. Med., 339(8): 504-10 1998; Van Damme et al., Lancet.,360(9338): 971-7 2002). In fact, it has been demonstrated that N-9actually increases the risk of genital inflammation (Stafford et al., J.Acquir Immune Defic Syndr. Hum. Retrovirol. 17(4): 327-31 1998), urinarytract infections (Fihn et al., Am. J. Epidemiol, 144(5): 512-20 1996),vulvovaginal candidiasis (Geiger and Foxman, Epidemiology., 7(2): 182-71996) and genital ulcers (Feldblum, Genitourin Med., 72(6): 451-2 1996).A recent study (Fichorova et al., J. Infect. Dis., 184(4): 418-28 2001)reveals that N-9 is cytotoxic for the vaginal epithelium and induces therelease of proinflammatory cytokines which, in turn, recruit immunecells that are targets for HIV, thus facilitating its tissue invasion.

For these reasons, there is a continuing need to develop a topicalformulation that is harmless to mucosae and the mucosal microflora,effective against HIV and other STD pathogens such as Herpes simplexvirus (HSV), Cytomegalovirus (CMV), Neisseria gonorrhoeae (NG), andChlamydia trachomatis (CT), and at the same time, offers contraceptiveprotection.

SUMMARY OF THE INVENTION

It has been discovered that compositions that include suramin or aderivative thereof are effective as a contraceptive and in inhibitingtransmission of sexually transmitted diseases. Accordingly, methods ofinhibiting transmission of sexually transmitted diseases and methods ofcontraception are provided. Compositions that include suramin or aderivative thereof, and devices coated or impregnated with suchcompositions are also provided herein.

In a first aspect of the invention, methods of inhibiting transmissionof sexually transmitted diseases are provided. In one form, a methodincludes topically, preferably vaginally, applying suramin or aderivative thereof. In certain forms of the invention, suramin may beco-administered in a composition with one or more antimicrobial agents.

In a second aspect of the invention, methods of contraception areprovided. In one embodiment, a method includes administering to thevagina an amount of suramin or a derivative thereof effective to inhibitsperm-egg fertilization, and thus prevent pregnancy. In certainembodiments, suramin may be co-administered with one or moreantimicrobial agents and/or sperm-function inhibitors.

In a third aspect of the invention, topical compositions are providedthat may be advantageously used to inhibit transmission of sexuallytransmitted diseases and inhibit sperm-egg fertilization. In oneembodiment, a composition includes a pharmaceutically-acceptable carrierand an amount of a surfactant and either suramin or a derivative thereofeffective to inhibit transmission of sexually transmitted diseases andinhibit sperm-egg fertilization.

In a fourth aspect of the invention, devices for administering suraminto the vagina or uterus are provided. In one embodiment, a deviceincludes a solid support adapted to be inserted into the vagina. Thesupport is advantageously impregnated with or coated with a compositionthat includes a surfactant and either suramin or a derivative thereof.

In a fifth aspect of the invention, methods for simultaneouslyinhibiting sexually transmitted infections and inhibiting sperm-eggfertilization are provided. In one form, a method includes administeringto a female mammal intravaginally, to the cervix or to the uterus, acomposition that includes suramin in an amount effective to inhibitsexually transmitted infections and sperm-egg fertilization. Thesexually transmitted infections may be caused by a microorganism, suchas Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum,Haemophilus ducreyi, Calymmatobacterium granulomatis, Mycoplasmagenitalium, Ureaplasma urealyticum, HIV-1, HIV-2, HTLV-1, herpes simplexvirus type 1, herpes simplex virus type 2, Epstein-Barr virus,cytomegalovirus, human herpesvirus 6, varicella-zoster virus, humanpapillomaviruses, hepatitis A virus, hepatitis B virus, Trichomonavaginalis, and Candida albicans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the hexasodium salt of suramin, 8,8′-(carbonylbis(imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene)carbonylimino-))bis-1,3,5-naphthalenetrisulfonic acid.

FIG. 2 is a graph depicting sperm motility and viability as a functionof indicated concentrations of suramin and nonoxynol-9 as more fullydescribed in Example 1.

FIG. 3 is a bar graph showing the effect of suramin on human sperm zonabinding and hamster egg penetration as more fully described inExample 1. HZA, hemizona assay; HEPT, hamster egg penetration test.

FIG. 4 depicts a graph representing sperm hyaluronidase activity as afunction of suramin concentration as more fully described in Example 1.

FIG. 5 depicts the infectivity remaining in indicated cells as afunction of pre-determined suramin concentrations as more fullydescribed in Example 2. VBI-BaL, viral entry inhibition assay with themonocytotropic HIV-1 strain BaL; CTC, cell-to-cell transmission assay;VBI-IIIB, viral entry inhibition assay with the lymphocytotropic HIV-1strain IIIB; API, active pharmaceutical ingredient (suramin drugsubstance).

FIG. 6 is a graph depicting inhibition of epithelial cell transmissionof HIV-1 by suramin in the presence and absence of mucin as more fullydescribed in Example 2.

FIG. 7 is a graph showing the anti-herpes activity and cytotoxicity ofsuramin as more fully described in Example 2.

FIG. 8 depicts a bar graph representing inhibition of cytomegalovirusbinding to fibroblasts as a function of the concentration of suramin asmore fully described in Example 2.

FIG. 9 depicts a graph showing the titer of Chlamydia trachomatis aftertreatment with various concentrations of suramin as more fully describedin Example 2.

FIG. 10 is a bar graph depicting growth of Neisseria gonorrhoeae as afunction of indicated concentrations of suramin determined by the N.gonorrhoeae (MSIIA) multiplication (growth) assay as more fullydescribed in Example 2.

FIG. 11 is a bar graph showing vaginal cell viability as a function ofindicated concentrations of suramin as more fully described in Example3.

FIG. 12 is a bar graph showing the concentration of indicatedinterleukins from human vaginal cell supernatants as a function ofconcentrations of suramin and nonoxynol 9 (N-9). Human vaginal (VK-2)cells were cultured for 6 hours in the presence or absence of theindicated compositions as more fully described in Example 3. Interleukin(EL) concentrations were determined in the cell supernatants. Resultsare expressed in EL concentration (pg/mL) normalized by percentage ofcell viability.

FIG. 13 is a graph showing the effect of suramin on growth ofLactobacillus as more fully described in Example 3. r² (coefficient ofcorrelation)=0.996. TD, doubling time.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been found that suramin and derivatives thereof in a topicalformulation have a combined effectiveness to inhibit transmission of STDinfections and reduce the probability of sperm-egg fertilization (i.e.,fertilization of an egg by sperm). Applicants have found that thetopical administration of suramin and derivatives thereof is effectiveto inhibit transmission of STDs and prevent pregnancy.

In a first aspect of the invention, methods of inhibiting transmissionof sexually transmitted diseases are provided. In one form, a methodincludes topically applying or otherwise administering suramin or aderivative thereof, typically to a site of infection.

Suramin is a polysulfonated naphthyl urea having the chemical name8,8′-(carbonylbis(imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene)carbonylimino))bis-1,3,5-naphthalene trisulfonic acid. The structure ofthe hexasodium salt of suramin is depicted in FIG. 1. Suramin iscommercially available and is also known under the following chemicaland trade names: Antrypol, Bayer 205, Fourneau 309, Germanin, Moranyl,Naganol, Naganin, Suramin, Naphuride Sodium. Suramin has been previouslyshown to inhibit in vivo activities of various growth factors andautoimmune and allergenic diseases (U.S. Pat. No. 5,158,940), possesspotent reverse transcriptase (RT) inhibitory activity (Jentsch et al.,J. Gen. Virol., 68: 2183-2192 1987), and anti-proliferative activity(Nakajima et al., J. Biol. Chem., 266(15): 9661-6 1991).

As used herein, “suramin” shall include both suramin andpharmaceutically acceptable salts thereof that are effective ininhibiting STD infections and inhibiting sperm-egg fertilization.Pharmaceutically acceptable salts, include, for example, alkaline metal,alkaline earth metal, other non-toxic metals, ammonium and substitutedammonium salts such as, but not limited to, the sodium, potassium,lithium, calcium, magnesium, aluminum, zinc, ammonium, trimethylammonium, triethyl ammonium, tetrabutyl ammonium, pyridinium andsubstituted pyridinium salts. Preferably, a hexasodium salt of suraminis employed.

“Derivatives” (analogues) of suramin are known in the art and includethose described, for example, by Jentsch et al., J. Gen. Virol., 68:2183-2192 (1987), and U.S. Pat. Nos. 5,173,509 and 6,121,320, which areboth herein incorporated by reference in their entirety. Other knownsuramin derivatives are described, for example, in U.S. Pat. No.6,121,320, which describes NF110, NF032, NF201, NF023, and NF103derivatives of suramin, and in Firsching-Hauck et al., Anticancer Drugs11 (2): 69-77 (2000) and Gagliardi et al., Cancer Chemother. Pharmacol.41(2): 117-24 (1998). The derivatives can be synthesized by methodsknown to the skilled artisan, including by the methods described in, forexample, Nickel P et al., Arzneim.-Forsch. 36, 1153-1157 (1986).

It has unexpectedly been found that combining suramin or a derivativethereof with other active agents described herein leads to synergisticinteractions. Accordingly, in yet another embodiment of the invention,the method described herein for inhibiting transmission of sexuallytransmitted diseases includes topically applying compositions thatinclude suramin and one or more active agents. As defined herein, anactive agent includes an antimicrobial agent, or other agent thatdisplays anti-STD pathogen activity. The active agent may also be asperm-function inhibitor that has the ability to inhibit the function ofsperm, to otherwise inhibit fertilization of an egg by sperm and/or tootherwise prevent pregnancy, such as by killing and/or functionallyinactivating sperm or by other effects on the activity of the sperm. Theactive agent may have at least dual functions, such as acting as asperm-function inhibitor and as an antimicrobial agent.

The antimicrobial agent may be active against algae, bacteria, fungi,parasites (helminths, protozoa), viruses, and subviral agents.Accordingly, the antimicrobial agent may be an antibacterial,antifungal, antiviral, antiparasitic, and an antiprotozoal agent. Theantimicrobial agent is preferably active against infectious diseases,such as sexually-transmitted diseases. Examples of microorganisms thatcause such diseases (and the diseases caused by such microorganisms)include Neisseria gonorrhoeae (gonorrhea); Chlamydia trachomatis(chlamydia, lymphogranuloma venereum); Treponema pallidum (syphilis);Haemophilus ducreyi (chancroid); Calymmatobacterium granulomatis(donovanosis), Mycoplasma genitalium, Ureaplasma urealyticum(mycoplasmas); human immunodeficiency virus HIV-1 and HIV-2 (HIV, AIDS);HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virus type 1 andtype 2 (HSV-1 and HSV-2); Epstein-Barr virus; cytomegalovirus; humanherpesvirus 6; varicella-zoster virus; human papillomaviruses (genitalwarts); hepatitis A virus, hepatitis B virus (viral hepatitis);Trichomona vaginalis (trichomoniasis); and yeasts, such as Candidaalbicans (vulvovaginal candidiasis). The antimicrobial agent may also beactive against other diseases that are transmitted by contact withbodily fluids that may also be transmissible by sexual contact and arecapable of being prevented by administration of the compositionsaccording to this invention. Accordingly, the phrase, “sexuallytransmitted diseases (STDs),” is to be interpreted herein as includingany disease that is capable of being transmitted in the course of sexualcontact, whether or not the genital organs are the site of the resultingpathology.

Suitable antiviral agents include, for example, virus-inactivatingagents such as the nonioinic, anionic and cationic surfactants discussedherein, and C31 G (amine oxide and alkyl betaine), polybiguanides,docosanol, acylcarnitine analogs, octyl glycerol, and antimicrobialpeptides such as magainins, gramicidins, protegrins, and retrocyclins.Mild surfactants may advantageously be used as antiviral agents in thecompositions described herein. In one embodiment, the suramincomposition for inhibiting STD infection includes an antiviralsurfactant that does not inhibit the STD-causing microorganism throughthe same mechanism as suramin. More preferably, the antiviral surfactantis sorbitan monolaurate. Other antiviral agents that may advantageouslybe utilized in the compositions described herein include nucleotide ornucleoside analogs, such as tenofovir, acyclovir, amantadine,didanosine, foscarnet, ganciclovir, ribavirin, vidarabine, zalcitabine,and zidovudine. Further antiviral agents that may be used includenon-nucleoside reverse transcriptase inhibitors, such as UC-781(thiocarboxanilide), pyridinones, TIBO, nevaripine, delavirdine,calanolide A, capravirine and efavirenz. From these reversetranscriptase inhibitors, agents and their analogs that have shown poororal bioavailability are especially suitable for vaginal administration,in combination with suramin, to prevent sexual transmission of HIV.Other antiviral agents that may be used in combination with suramin arethose in the category of HIV entry blockers, such as cyanovirin-N,clyclodextrins, carregeenans, sulfated or sulfonated polymers, mandelicacid condensation polymers, monoclonal antibodies, chemokine receptorantagonists such as TAK-779, SCH-C/D, and AMD-3100, and fusioninhibitors such as T-20 and 1249.

Suitable antibacterial agents include antibiotics, such asaminoglycosides, cephalosporins, including first, second and thirdgeneration cephalosporins; macrolides, including erythromycins,penicillins, including natural penicillins, penicillinase-resistantpenicillins, aminopenicillins, extended spectrum penicillins;sulfonamides, tetracyclines, fluoroquinolones, metronidazole and urinarytract antiseptics.

Suitable antifungal agents include amphotericin B, nystatin,griseofulvin, flucytosine, fluconazole, potassium iodide, intraconazole,clortrimazole, miconazole, ketoconazole, and tolnaftate.

Suitable antiprotozoal agents include antimalarial agents, such aschloroquine, primaquine, pyrimethamine, quinine, fansidar, andmefloquine; amebicides, such as dioloxamide, emetine, iodoquinol,metronidazole, paromomycine and quinacrine; pentamidine isethionate,atovaquone, and eflornithine.

The suramin compositions of the present invention according to themethods described herein are administered or otherwise applied bytopically delivering the composition, typically to a site of infection.The site of infection may be one where an infection is already present(an actual site of infection) or where an infection is likely to occur(a potential site of site of infection in or on an uninfectedindividual). Accordingly, the compositions may be topically delivered tothe vulva, including the vaginal cavity, the penis and the ano-rectaland buccal cavities by contacting the skin or mucosae of the intendedsite or surrounding the intended site. The mucosal or skin surface mayfurther include the perianal, and the lining of the anus. For example,in the case of inhibiting STD-infections, the suramin compositions ofthe present invention may be administered by being contacted with anypotential or actual sites of infection, including the vaginal,ano-rectal or buccal cavities to prevent STD infection during intimateactivity. When administered to the vaginal cavity, the compositions mayalso be applied to any portion of the uterus, including inside theuterus and on the cervix, including the mucosa and/or lining of theendo- and ecto-cervix. Moreover, when formulated as a lubricant, thecompositions can be applied to external genitalia and internal mucosalsurfaces to reduce microtrauma resulting from inadequate lubrication andwill also prevent transmission of viable STD pathogens throughtraumatized, diseased or healthy skin or mucosa.

A dose of the pharmaceutical composition is preferably made up of one ormore pharmaceutical dosage units. The selected dose may be administeredto a mammal, for example, a human, by any known method of administeringthe dose, including the methods described herein, for example, as asuppository or gel to be applied to the vagina, rectum or uterus. Forexample, the suramin compositions may be delivered intravaginally byapplying as a lubricant, for example, on a device, including a sponge,cervical cap, tampon, diaphragm, or intrauterine device or by applyingthe composition as a suppository, douche, ovule, gel, or othercontrolled delivery device. Although placing the compositions on acondom can result in transfer of some of the compositions to a mucosalsurface and provide a degree of protection for such surfaces, theprimary benefits of such an application include protection of the condomwearer. The suramin compositions may be applied to any portion of theuterus by an intrauterine delivery device, such as those intrauterinedevices (IUDs) known to those skilled in the art. Applicators known tothe art, such as those currently used commercially to deliverspermicidal gels or anti-yeast compounds, may also be used to deliverthe compositions intravaginally.

A STD-infection inhibitory or otherwise preventative or effective amountof the suramin composition is typically administered. This amount isintended to mean that amount of suramin, when administered to a mammalin need thereof, that is sufficient to effect inhibition of transmissionof STD infections. In other words, the dose is that effective to preventSTD infection at the site of entry or to prevent the replication of theSTD-causing microorganism. In the case of inhibiting transmission of STDinfections, the effective amount of suramin may vary depending uponfactors such as the STD-causing microorganism intended to be inhibitedby blocking its entry or ceasing its replication at the site ofinfection. For example, the suramin compositions of the presentinvention are suitably formulated to inhibit human immunodeficiencyvirus (HIV) (including, but not limited to, HIV-1 and HIV-2), Herpessimplex virus (HSV), Cytomegalovirus (CMV), Neisseria gonorrhoeae (NG),and Chlamydia trachomatis (CT). Preferably, the effective amount ofsuramin is that which is effective to inhibit transmission of any of thepotential STD-causing microbes known to the art and described herein.Although these amounts may vary, suramin is typically applied in anamount of from about 0.1% (1 mg of suramin per 1 gram of formulation) toabout 30% (300 mg/g), preferably about 1% (10 mg/g) to about 5% (50mg/g); or in a way in which the delivery system releases enough suraminto maintain these percent concentrations in weight over volume. Whenpresent in a composition with an antimicrobial agent, the amount ofsuramin in the composition applied will also vary with respect to thespecific antimicrobial agent used and may be readily determined by theskilled artisan. The amount of the antimicrobial agent in thecomposition will vary depending on, for example, the nature of thedisease involved and the amount of suramin in the composition and mayalso be readily determined by the skilled artisan. These amounts may beadministered to an individual with a STD or to an individual who intendsto have sexual contact with an individual with a STD.

“Inhibiting” or “inhibit” as referenced in the methods of the presentinvention is intended to mean at least the reduction in incidence orprevalence of the occurrence of the specified activity relative to anuntreated individual. These terms may include the prophylactic orpreventative treatment in a mammal or the reduction in the incidence ofa condition relative to untreated mammals. The “inhibition” oftransmitting STD infection refers to the reduced transmission of STDinfection as a result of treatment of the individual prior to, orimmediately after, intimate contact relative to untreated individuals.In such case, and not being limited by any particular mechanism ofaction, the inhibition of transmitting STDs may be caused byneutralizing the microorganism causing the STD infection at the site ofinfection or by preventing the replication of the microorganism at thesite of infection. The inhibition of the STD infection is between atleast two people in sufficient contact with each other to, contract theSTD infection. For example, the inhibition of the STD infection can bebetween two persons involved in sexual contact or between a mother andchild (vertical transmission).

A wide variety of STD infections may be inhibited according to themethods of the present invention. For example, suramin, and thecompositions described herein, can be used against microorganisms withinthe categories of algae, bacteria, fungi, parasites (helminths,protozoa), viruses, and subviral agents. For example, suramincompositions may be applied according to the methods of the presentinvention to inhibit transmission of diseases caused by variousmicroorganisms, including Neisseria gonorrhoeae (gonorrhea); Chlamydiatrachomatis (chlamydia, lymphogranuloma venereum); Treponema pallidum(syphilis); Haemophilus ducreyi (chancroid); Calymmatobacteriumgranulomatis (donovanosis); Mycoplasma genitalium, Ureaplasmaurealyticum (mycoplasmas); human immunodeficiency virus HIV-1 and HIV-2(HIV, AIDS); HTLV-1 (T-lymphotrophic virus type 1); herpes simplex virustype 1 and type 2 (HSV-1 and HSV-2); Epstein-Barr virus;cytomegalovirus; human herpesvirus 6; varicella-zoster virus; humanpapillomaviruses (genital warts); hepatitis A virus, hepatitis B virus(viral hepatitis); Trichomona vaginalis (trichomoniasis); and yeasts,such as Candida albicans (vulvovaginal candidiasis). The diseases causedby such microorganisms are shown in parenthesis above.

In yet other forms of the invention, the STD infections may be caused bybacteria. Examples of such bacteria include Neisseria gonorrhoeae,Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi,Calymmatobacterium granulomatis, Mycoplasma genitalium, and Ureaplasmaurealyticum. In yet other embodiments, the STD infections may be causedby a virus, including HIV-1, HIV-2, HTLV-1, herpes simplex virus type 1,herpes simplex virus type 2, Epstein-Barr virus, cytomegalovirus, humanherpesvirus 6, varicella-zoster virus, human papillomaviruses, hepatitisA virus, and hepatitis B virus. In other embodiments of the invention,the STD infections may be caused by a virus, such as HTLV-1,Epstein-Barr virus, varicella-zoster virus, human papillomaviruses,hepatitis A virus, and hepatitis B virus. In further embodiments, theSTD infection may be caused by Candida albicans and Trichomonavaginalis.

In yet another aspect of the invention, methods of contraception, orotherwise preventing pregnancy, are provided. In one form, a methodincludes administering to the vagina an amount of suramin or derivativethereof effective to inhibit sperm-egg fertilization and otherwiseprevent pregnancy. In other embodiments of the invention, the suramin orderivative thereof may be combined with a different sperm-functioninhibitor, as such a combination has been found herein to exertsynergistic effects with respect to contraception. Compositions thatinclude suramin and a derivative thereof, optionally in combination withthe active agents described herein, are also envisioned for use in themethods described herein.

The sperm-function inhibitor that increases the contraceptive propertiesof suramin may be selected from, for example, surfactants, includingnonionic surfactants, cationic surfactants, and anionic surfactants;spermicides, such as nonoxynol-9(α-(4-Nonylphenyl)-ω-hydroxynona(oxyethylene); other sperm-inactivatorssuch as sulfated or sulfonated polymers such as polystyrene sulfonate,mandelic acid condensation polymers, cyclodextrins; antimicrobialpeptides such as gramicidins, magainins, indolicidin, and melittin; andacid-buffering compositions, such as BufferGel and AcidForm. Nonionicsurfactants include, for example, sorbitan monolaurate,nonylphenoxypolyethoxy ethanol, p-diisobutyphenoxypolyethoxy ethanol,polyoxyethylene (10) oleyl ether and onyx-ol. Suitable anionicsurfactants include, without limitation, sodium alkyl sulfonates and thesodium alkylbenzene sulfonates. Cationic surfactants include, forexample, the quaternary ammonium surfactants, such as cetyl pyrimidiniumchloride and benzalkonium chlorides. Zwitterionic surfactants such asacylcarnitine analogs and C31G are especially suitable for their mildskin and mucosal irritation properties.

In the case of inhibiting sperm-oocyte fertilization, or otherwisepreventing pregnancy, the suramin compositions of the present inventionare intravaginally applied either directly or indirectly. For example,the suramin compositions may be delivered intravaginally by applying asa lubricant, for example, on a condom or other device, including asponge, cervical cap, tampon, diaphragm, or intrauterine device or byapplying the composition as a suppository, douche, ovule, gel, or othercontrolled delivery device. The suramin compositions may be applied toany portion of the uterus by an intrauterine delivery device, such asthose intrauterine devices (IUDs) known to those skilled in the art.Applicators known to the art, such as those currently used commerciallyto deliver spermicidal gels or anti-yeast compounds, may also be used todeliver the compositions.

An effective amount of the suramin compositions is typicallyadministered. This effective amount, in the context of the methods ofcontraception described herein, is intended to mean that amount ofsuramin, when administered to a mammal in need thereof, sufficient toeffect inhibition of sperm-egg fertilization and embryo formation. Inthe case of its contraceptive properties, the effective amount ofsuramin is that amount effective to decrease the possibility ofsperm-egg fertilization, either by blocking entry of sperm into the egg,inhibiting sperm-fertilizing capabilities or by other methods. Thisamount of suramin may be readily determined by one of skill in the art.For example, the suramin may be present, in an effective amount,preferably in an amount of from about 1 to about 300 mg per gram ofcomposition, preferably about 5 to about 100 mg/g, or in the range of0.0001-90%, preferably about 0.01 to about 30% or about 0.5% to about10% by weight of the composition. Higher and lower amounts may also beeffectively employed in the practices of this invention.

Moreover, the “inhibition” of sperm-egg fertilization refers to thereduced occurrence of conception, i.e., sperm-egg fertilization,resulting in pregnancy relative to untreated individuals. In such case,the suramin may act in a number of different ways. For example, it mayinhibit fertilization by blocking sperm receptors to the zona pellucida.Alternatively, it may inhibit hyaluronidase or other sperm-enzymeinteractions required for fertilization. It may also agglutinate sperm,impeding normal ascent or transport through the female genital tract.Whatever the mechanism, and not being limited by any particular theoryof the mechanism of action of the compositions described herein, theinhibition of sperm-egg fertilization results in an effectivecontraceptive property for the suramin topical pharmaceuticalcompositions of the present invention.

The compositions used in the methods described herein may include otheragents that do not negatively impact or otherwise affect themicrobicidal and/or contraceptive effectiveness of the components of thecomposition, including antimicrobial agents, sperm-function inhibitors,suramin or derivatives of suramin. For example, solid, liquid or amixture of solid and liquid pharmaceutically acceptable carriers,diluents, vehicles, or excipients may be employed in the pharmaceuticalcompositions. Suitable physiologically acceptable, substantially inertcarriers include water, a polyethylene glycol, mineral oil orpetrolatum, propylene glycol, hydroxyethylcellulose, carboxymethylcellulose, cellulosic derivatives, polycarboxylic acids, linkedpolyacrylic acids, such as carbopols; and other polymers such aspoly(lysine), poly(glutamic acid), poly(maleic acid), poly(lactic acid),thermal polyaspartate, and aliphatic-aromatic resin; glycerin, starch,lactose, calcium sulphate dihydrate, terra alba, sucrose, talc, gelatin,pectin, acacia, magnesium stearate, stearic acid, syrup, peanut oil,olive oil, saline solution, and the like.

The pharmaceutical compositions described herein useful in the methodsof the present invention may further include diluents, fillers, bindingagents, moisturizing agents, preservatives, acids, and other elementsknown to those skilled in the art. For example, suitable preservativesare well known in the art, and include, for example, methyl paraben,propyl paraben, butyl paraben, benzoic acid and benzyl alcohol.

The compositions used in the methods of the invention may be employed inany form suitable for topical application. For example, the compositionsof this invention could be in various forms known to the art, includingliquid form or in lotion form, either oil-in-water or water-in-oilemulsions, in aqueous gel compositions, in the form of foams, films,sprays, ointments, pessary, suppository, capsules, tablets, jellies,creams, liposomes or in other forms embedded in a matrix for the slow orcontrolled release of the biologically active material to the skin orsurface onto which it has been applied or in contact. Preferably, thecompositions of the present invention are aqueous compositions. Mostpreferably, the compositions are aqueous gel compositions.

In yet another aspect of the invention, methods for simultaneouslyinhibiting sexually transmitted diseases or infections and inhibitingsperm-egg fertilization are provided. The suramin compositions of thepresent invention are effective for simultaneously inhibiting mucosalentry of STD-causing microorganisms, including HIV, Herpes Simplexvirus, Cytomegalovirus, Chlamydia trachomatis and Neisseria gonorrhoeae,as well as inhibiting sperm-egg fertilization in a female mammal. Insuch a case, the suramin composition is delivered intravaginally to afemale mammal in a microbicidal contraceptive amount sufficient toinhibit sperm-egg fertilization in the female mammal and to inhibit STDsor infections. The inhibition of STD may occur by, for example,preventing STD-causing microorganisms from entering the cervico-vaginalmucosa and/or preventing such microorganisms to replicate and grow inthe female mammal.

Reference will now be made to specific examples illustrating themethods, compositions and devices above. It is to be understood that theexamples are provided to illustrate preferred embodiments and that nolimitation of the scope of the invention is intended thereby.

Example 1 Anti-Sperm Activity Sperm Immobilization

The Sander Cramer test was used to determine the effect on spermimmobilization as described in Sander F V and Cramer S D., Hum. Fertil.6: 134-153 (1941). This test may be used to evaluate thesperm-immobilizing effectiveness of contraceptive compositions. Serialdilutions of each test composition were added to semen adjusted to adesignated number, e.g., 60 million of motile sperm per milliliter atroom temperature (i.e., 25° C.). The end point is the greatest dilutionat which all of the sperm are immobilized within 20 seconds. Results areexpressed as minimum effective concentration in milligrams permilliliter.

Suramin showed no significant sperm-immobilizing activity at 20 mg/mL.Additionally, all spermatozoa from samples were motile when theexperiment was conducted with 10 and 5 mg/mL suramin sodium. In moredetailed dose-response studies using 1 log-fold concentrations (1-10,000μg/mL) or 2-fold serial dilutions (5-0.07 mM) and sperm progressivemotility and membrane integrity as endpoints, suramin sodium did notshow significant differences with its solvent control, i.e., it inducedno alteration of sperm progressive movement and viability (FIG. 2).Conversely, the commercial spermicide nonoxynol-9 (N-9), run inparallel, displayed its known sperm-immobilizing effects. In atime-dependent study at suboptimal doses, unlike N-9, suramin sodiumshowed no alteration of sperm motility even after prolonged incubationtime (e.g., 30 min.). At 1 mM, no significant changes in sperm motionparameters were detected by CASA (Computer-Assisted Semen Analysis) asdescribed, for example, in WHO laboratory manual for the examination ofhuman semen and sperm-cervical mucus interaction, 4th ed., WHOOrganization, Cambridge University Press, pp. 30-33, 1999. Even athigher concentrations (e.g., 7.5 mg/mL), sperm motion characteristicsdid not appear to be significantly different from those of the control(solvent-treated) sperm.

Cervical Mucus Penetration Effects

Bovine cervical mucus was obtained from Humagen (Charlottesville, Va.)in the form of prepackaged flat capillary tubes (Penetrak®), kept frozenand thawed right prior to the experiment. The Modified One End Test(MOET) [(described in Doncel G. F., in “Barrier Contraceptives”,Wiley-Liss, Mauck C. K. et al. eds., pp. 147-162 (1994)] was used todetermine the effect of compounds, such as suramin sodium describedherein, on sperm penetration in cervical mucus. Each of the testcompositions containing the compound to be tested were diluted in asaline solution, i.e., at 9 grams of NaCl per liter of water. Tubes ofcervical mucus were broken open. The open end was placed in a containercontaining the test compound in saline. The test compound was allowed todiffuse for 30 minutes through the tube. A semen sample, obtained fromnormal healthy donors, was then diluted with a buffer solution to 60million motile sperm per milliliter and mixed with the test compoundsample (i.e., suramin sodium). The tube containing the test compoundsample was then re-inserted into the container containing the mixedsolution and stored in an incubator at 37° C. in an atmosphere of 5percent carbon dioxide in air for 60 minutes. The container and tubewere then removed from the incubator and the tube was visually analyzedunder a microscope for the migration of motile vanguard sperm throughthe tube. The results are expressed as percentage of migration ascompared to control samples. In the control samples, the tubes wereincubated with saline containing no compound.

The Simultaneous One End Test (SOET) was used to detect the quickblocking effects of the test compounds particularly exerted throughsperm motility alterations and is described in Doncel G. F., in “BarrierContraceptives”, Wiley-Liss, Mauck C. K. et al. eds., pp. 147-162(1994)]. The SOET is similar to the MOET except that the solutioncontaining the test compound was mixed with the semen sample and thenone end of the capillary tube containing bovine cervical mucus wasinserted into the mixture of the test compound and semen sample andstored in an incubator at 37° C. in an atmosphere of 5% carbon dioxidein air for 60 minutes. Penetration length of vanguard motile sperm wasrecorded and the results are expressed as percentage of migration ascompared to control samples, i.e., saline containing no compound. In theSOET, if not impeded by the test compound, the sperm have the ability tomigrate into the tube immediately after contact.

A dose-dependent study showed only minor inhibition of sperm penetrationwhen suramin sodium was tested at 50 mg/mL (5%) in the MOET test. Nosignificant sperm impediment was found at 25 mg/mL or below. At 5%,dextran sulfate showed more inhibition than suramin sodium. Forcomparison purposes, N-9 completely blocked sperm penetration at 300μg/mL (0.03%). KY Jelly® (KY), a commercial vaginal lubricant andsuramin sodium in KY showed the same degree of inhibition (˜40%), whichwas significantly different from suramin sodium (˜4%) alone at the sameconcentration. No major sperm clumping was observed at any concentration(Table 1).

TABLE 1 Cervical Mucus Blocking Activity of Suramin. MOET CompoundSolvent Dilution % CTL n Suramin 0.9% NaCl 50 mg/ml 72.3 ± 7.1 10 25mg/ml 95.9 ± 1.4 10 12.5 mg/ml 94.9 ± 2.2 5 6.25 mg/ml 96.1 ± 0.9 5Dextran Sulfate 0.9% NaCl 50 mg/ml 56.6 ± 8.9 10 25 mg/ml 86.8 ± 4.4 1012.5 mg/ml 82.4 ± 5.2 5 6.25 mg/ml 93.7 ± 1.7 5 N-9 0.9% NaCl 300 ug/ml 0.7 ± 0.5 10 30 ug/ml 48.6 ± 6.9 10 Suramin-KY dH2O 25.5 mg/ml-25% 64.1± 7.6 10 KY 0.9% NaCl 25% 61.6 ± 6.9 10

Viability of Sperm

Viability of sperm was assessed by a sperm membrane integrity evaluationas described in Jeyendran R. S. et al., J. Reprod. Fertil., 70: 219-28(1984). Aliquots of sperm samples were incubated with suramin or N-9test compounds. The reaction was terminated by the addition of 1.5 mLHam's F10. Sperm were centrifuged and the pellet was resuspended in 200μL of medium. Sperm viability was assessed by placing 100 μL of thesperm suspension in 900 μL of hypo-osmotic swelling test (HOST) mediumfor 30 min. Coiled sperm tails are a reflection of intact sperm membranepermeability and indicate viable sperm. A minimum of 200 cells weremicroscopically assessed from each sample.

The results of the sperm membrane integrity evaluation are shown in FIG.2. No significant differences were observed in sperm viability whensuramin-treated samples (10-0.001 mg/mL) were compared tosolvent-treated controls. After a fifteen minute co-incubation, motilityand viability showed very similar response patterns in both dose andtime-dependent experiments, indicating that suramin is not a spermicidalcompound.

Human Sperm-Zona Binding

The hemizona assay, as described in Burkman L. J., et al., Fertil.Steril., 49: 688-697 (1988), was used to measure the ability of sperm toundergo capacitation and bind to the zona pellucida of an oocyte.Briefly, in this assay, motile normal sperm were separated in media withbovine serum albumin, which triggers capacitation. Human motile spermwere separated using a swim-up technique and incubated with 1 mM suraminor solvent-control medium for 15 minutes in 100 μL medium dropletscovered with mineral oil, at 37° C., 5% CO₂. Sperm were then incubatedwith dead oocytes which are surrounded by the zona pellucida, anacellular coating of oocytes. Bisected hemizonae corresponding to oneoocyte were placed each in the test (suramin) and control (solvent)droplets and incubated for an additional 4 hours. Sperm-hemizonaecomplexes were washed extensively and capacitated spermatozoa bound tothe outer surface of the zonae and were counted under an invertedmicroscope. The Hemizona Index (HZI) was calculated by dividing thenumber of suramin-treated sperm bound to one hemizona by the number ofuntreated (control) sperm bound to the other hemizona.

The results of the hemizona assay are shown in FIG. 3. It was found thatsuramin was a potent sperm-zona binding inhibitor, producing 92%inhibition of human sperm binding at 1 mM (HZI=0.08±0.01, n=9) (FIG. 3).Inhibition may be mediated by blocking zona receptors at thesperm-surface level and/or interfering with signal transduction.

Sperm-Oolemma Binding and Penetration

The hamster-egg penetration test (HEPT) was used to predict thefertilizing ability of human sperm by evaluating the sperm'sooctye-penetration capability as described in Yanagimachi R. et al.,Biol. Reprod., 15: 471-6 (1976). The zona pellucida of hamster eggs wasdissolved by treatment with a protease, and human sperm weresubsequently added to the zona free eggs. Sperm binding and penetrationwere assessed under a standard microscope (60× magnification) afterplacing the sperm-egg complexes onto polylysine-coated glass slides andcovered them with 22×22 mm glass coverslips. The ability of the sperm tobind and enter the egg was scored by counting the sperm nuclei lyingwithin the egg cytoplasm. The effect of the test compound was determinedby comparing the number of sperm bound to the surface of the eggs andthe percentage of penetrated eggs.

The results of the HEPT assay using suramin sodium as the test compoundare shown in FIG. 3. Suramin sodium showed decreased sperm binding tohamster's zona-free oocytes (˜75% inhibition) and completely abolishedsperm penetration/fertilization of such eggs in-vitro (n=28 oocytes).

Sperm Hyaluronidase Activity

A hyaluronic acid hydrolysis assay was used to assess the hyaluronidaseinhibition properties of suramin sodium. Hyaluronidase is a criticalsperm enzyme involved in cumulus penetration. Inhibition of this enzymerenders sperm incapable of transversing the egg vestments, thus impedingsperm-oocyte interaction and, ultimately, fertilization.

Hyaluronidase activity was quantitatively determined by measuring theextent of hyaluronic acid hydrolysis (i.e., the concentration ofN-acetylglucosamine-reactive material formed from enzyme activity).Reaction mixtures containing the following were prepared: test compound(variable concentrations of suramin sodium); 0.1 M sodium acetate; 0.15M sodium chloride; pH adjusted to 5.5; 7.2 units sheep testicularhyaluronidase (Sigma, Type III; H-2251) contained in an acetate buffer;0.3 mg/ml hyaluronic acid (Sigma, from bovine vitreous humor; H-7630).The enzyme was preincubated with the test agent (1 mg/ml for screeningpurposes) for 10 minutes before starting the reaction by the addition ofhyaluronic acid. The enzyme reaction was determined using the method ofAronson and Davidson (J. Biol. Chem. 241 437-40, 1967). The reactionmixture was incubated for 30 minutes at room temperature. The reactionproduct is determined calorimetrically withβ3-dimethylaminobenzaldeh-yde (Reissig et al., J. Biol. Chem. 217959-966, 1965) by measuring the absorbance at 545 nm. Compounds whichshow no inhibition at the screening concentration of 1 mg/ml areconsidered to be inactive. If the test agent shows inhibition at thescreening concentration, a dose-response curve is generated from whichIC₅₀ values could be determined using curve-fitting software.

The results of the Hyaluronidase activity assay tested with suraminsodium are shown in FIG. 4. Suramin sodium proved to be a potent andirreversible inhibitor of hyaluronidase displaying an IC₅₀=22 μg/mL. Onemg/mL induced 100% inhibition (FIG. 4).

Acrosome Reaction

The acrosome reaction ionophore challenge test (ARIC) was used, asdescribed in Cummins J. M., et al., J. Androl., 12: 98-103 (1991), tomeasure the proportion of spermatozoa that respond to a calciumionophore (typically, A23187), developing an acrosome reaction. Morespecifically, this test was used to determine whether suramin sodiumaffects the ionophore-induced acrosome reaction of the sperm, thereforealtering the capacity of sperm to penetrate an egg. Results areexpressed in percent inhibition of control (untreated sperm) acrosomereactions. The results of the ARIC with suramin sodium are shown inTable. 2.

TABLE 2 Effect of Suramin on Human Sperm Acrosome ReactionCalcium-ionophore Percentage of treatment acrosome-reacted Compound (5μM A23187) Sperm n Suramin Sodium − 5.28 ± 1.93 6 Control − 5.39 ± 1.386 Suramin Sodium + 31.4 ± 15.1 6 Control + 29.8 ± 13.0 6

Referring to Table 2, suramin sodium appeared neither to stimulatespontaneous acrosome reaction (acrosomal loss) nor to block the Ca²⁺ionophore-induced reaction, as has been described for other sulfated andsulfonated compounds, especially polymers.

Rabbit Contraceptive Efficacy Trials

Semen was collected from New Zealand White (NZW) male rabbits with theaid of an artifical vagina, pooled and adjusted with a modified Tyrode'salbumin lactate pyruvate (TALP) medium to 50 million motile sperm permilliliter. Ovulation was induced in females by injection of 200 IU ofhuman chorionic gonadotropin (hCG) intravenously. Under the ex-vivo(in-vitro) mixing protocol, sperm were incubated with indicated suramintest compounds in a tube for 15 minutes at 37° C. prior to insemination(0.5 mL) as described in Anderson R. A., et al. J. Androl., 21: 862-875(2000). If the suramin composition showed contraceptive activityfollowing this protocol, it was then tested in a formulation which isapplied intravaginally (1 mL) 15-30 minutes before insemination.Sperm-compound contact, in this case, took place in vivo, inside thevagina. Insemination and administration of the compound was performedwith a flexible, 12 cm long polyurethane catheter. The main endpointsfor both protocols were the number of implantation sites counted 11 daysafter insemination and pregnancy rate. Such sites were counted by visualinspection of the tubes.

Ex Vivo Trial

Referring to Table 3, under an “ex-vivo mixing” format where sperm wereincubated in-vitro with medium with or w/o suramin sodium, 1 mM (1.4mg/mL) concentrations inhibited pregnancy rates (PR) by 86% (1/7) alsoreducing the number of implantation sites (IS) from 6.4±0.9 in controlsto 1 in the test group. A concentration of 0.1 mg/mL did notsignificantly reduce PR, although it slightly decreased IS from 7.7±1.2to 4.4±0.8. A five percent (5%) concentration completely preventedpregnancies (0/7). The results are shown in Table 3.

TABLE 3 Contraceptive efficacy of suramin sodium after ex-vivo spermmixing Suramin Implantation Concentration Number of Pregnant PregnancySites Compounds (mg/ml) Females/Total Rate (%) (Mean ± SD) Mediumcontrol 0 7/7 100 7.7 ± 1.2 Suramin 0.1 5/6 83 4.4 ± 0.8 1.4 1/7 14 1 500/7 0 N.A. Pooled rabbit sperm were mixed with suramin at variousconcentrations, in vitro, for 15 minutes, before insemination. Femaleswere induced to ovulate with hCG and then intravaginally inseminatedwith 50 million, treated or untreated motile sperm/mL (0.5 mL).Pregnancy and implantation sites were evaluated 11 dayspost-insemination.

In Vivo Trial

After intravaginal administration of 5% suramin sodium in KY Jelly® (KY)or carboxymethyl cellulose (CMC) followed by artificial insemination,there was a 75% reduction in PR (2/8). In order to verify a potentialadditive effect which could increase suramin's efficacy, a pilot studywas performed adding a mild non-ionic surfactant, sorbitan monolaurate,to the suramin sodium/KY formulation. PR for control, 2% sorbitan, 2%suramin sodium, and 2% sorbitan+2% suramin sodium were 100%, 100%, 44%,and 0%, respectively (Table 4). Another experiment combining 5% suraminwith 0.1% N-9 in KY also showed synergistic effects.

TABLE 4 Contraceptive Efficacy of Suramin Sodium After IntravaginalApplication and Cooperative Effects of Surfactants ConcentrationsImplantation of Actives Number of Pregnant Pregnancy Sites Compounds(mg/mL) Females/Total Rate (%) (Mean ± SD) KY Jelly ® 0 7/8 87.5 5.9 ±0.8 Suramin in KY 50 2/8 25 8.5 ± 0.5 Suramin in KY 20 4/9 44 5.0 ± 1.1Sorbiton ML in KY 20 4/4 100 3.0 ± 1.0 Suramin + Sorbitan ML in KY 20 +20 0/4 0 N.A. Control and test formulations were administeredintravaginally (1 mL) to females before induction of ovulation with hcG.Fifteen to thirty minutes later, the females were inseminated withnormal untreated pooled rabbit sperm (50 million motile sperm permilliliter 0.5 mL)

Referring to Table 5, a synergistic effect was clearly evident whensuramin was formulated at various concentrations, from 5% to 1.25%, in acarbopol-based vaginal lubricant gel, Replens®. Even the lowest dosestudied, 1.25%, was 100% contraceptive (PR=0%). The PR in the controlgroup (Replens® alone) was 100%.

TABLE 5 Contraceptive Efficacy of Suramin Formulations - Dose ResponseSuramin Implantation Concentration Number of Pregnant Pregnancy SitesCompounds (mg/mL) Females/Total Rate (%) (Mean ± SD) Replens ® (control)0 4/4 100 10.5 ± 1.73 5% suramin in Replens ® 50 0/4 0 N.A. 2.5% suraminin Replens ® 25 0/4 0 N.A. 1.25% suramin in Replens ® 12.5 0/4 0 N.A.Female rabbits were administered intravaginally with 0.75 mL of compoundand 15-30 minutes later inseminated with pooled rabbit sperm.Implantation sites were visualized 11 days after insemination

A summary of the anti-sperm and contraceptive activity of the compoundsof the present invention is provided in Table 6.

TABLE 6 Summary of anti-sperm and contraceptive activity of suramincompositions Type of Inhibition/ Activity Inactivation Assays PotencyComments Anti-Sperm Motility Sander Cramer None Highest concentration =(in-vitro) Motility 25 mg/mL CASA Time and dose-dependent exps.Computer-assisted motion analysis Viability HOST None Time- anddose-dependent exp. CM Penetration MOET Very low Tested formulated (5%)and SOET unformulated ZP binding HZA High 92% inhibition at 1 mM-Reversible Oolemma binding/ HEPT High 75% and 100% inhibition ofPenetration binding and penetration, respectively HyaluronidaseHyaluronic Acid High IC₅₀ = 22 μg/mL- Irreversible Hydrolysis AcrosomeReaction ARIC None Induction or inhibition of acrosome reactionContraception Rabbit pretreated sperm In-vitro mixing Moderate 100%inhibition at 5% in KY (in-vivo) Jelly ®; 86% inhibition at 0.1% (~1 mM)Rabbit inseminated Vaginal Moderate 75% inhibition at 5%; spermApplication 44% at 2% Vaginal High 100% inhibition at 2% suramin +Application 2% sorbitan mono laurate or 0.1% Combo N-9 Vaginal High 100%inhibition at 1.25% Application Replens ®

Example 2 Anti-Microbial Activity

Activity against HIV

Inhibition of HIV Infectivity:Virus Binding Inhibition Assay

VBIAIIIB: MT-2 cells, compounds and virus (HIV-1 IIIB) were mixedtogether in 96-well plates and incubated for 2 h at 37° C. At the end ofthis incubation period, the 96-well plates were centrifuged for 10minutes and approximately 175 μL of medium was removed using amultichannel pipettor and replaced with 175 μL of fresh media. Cultureswere then incubated at 37° C. for 6 days. Modulation of virus inducedcytopathic effects was measured by determining percent reduction inoptical density (O.D.) using an XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[phenylamino)carbonyl]-2H-tetrazoliumhydroxide) dye reduction assay. Virus-induced cytopathic effects andmechanical artifacts due to media removal were verified by microscopicobservations (Anderson R. A., et al., J. Androl., 21: 862-875 (2000)).The results of this assay are provided herein.

C-MAGI/Ba-L Assay

The C-MAGI/Ba-L assays were carried out with MAGI-CCR-5 cells. The cellline was maintained in DMEM medium containing 10% FBS, 100 U/mlpenicillin, 300 μg/mL glutamate and selection antibiotics as indicatedabove. Before assay, the cells were placed in 96 well flat tissueculture plates at 1×10⁴ cells per well, and incubated overnight in theabsence of antibiotics. The next day, medium was removed, and virus andcompound (100 μL virus (HIV_(Ba-L))+100 μL compound) were added. Thecultures were incubated for 2 hours to facilitate virus attachment.After this incubation, the medium is removed from the wells, replacedwith fresh compound-free medium, washed twice and finally incubated at37° C. for 2 days in fresh medium. Following incubation, the medium wasremoved and induction of β-galactosidase enzyme activity was detected bychemiluminescence. β-galactosidase activity was detected using achemiluminescence substrate, following the manufacture's instructions(Tropix). The TC₅₀ and IC₅₀ are determined using linear regression and atherapeutic index or antiviral index calculated. The results of thisassay are provided herein.

ME-180-Based Topical Microbicide Assay

ME-180 cervical epithelial cells were plated in the interior wells of a96-well flat bottom microtiter plate at a density of 5×10³ cells perwell and incubated overnight. Chronically infected H9 cells (H9-SK1)were treated with 200 μg/ml mitomycin C (Sigma) in complete medium forone hour, washed extensively and resuspended at 4×10⁵ per ml. Theconcentration of mitomycin C used resulted in the killing of thechronically infected cells within 48 hours treatment, allowingsufficient time for cell-cell transmission of virus to the ME-180 cellswhile assuring that the virus endpoint quantification would not includea contribution from the chronically infected cells. Antiviral compoundsand chronically infected cells (2×10⁴ cells) were added to each wellcontaining ME-180 cells and incubated for 6 hours. Followingco-cultivation, the monolayer was washed extensively and fresh mediumwas added. Medium was removed and fresh medium was added at 24 and 48hours post-infection to remove dead lymphocytes. On day 6post-infection, supernatant samples were removed and analyzed for viruscontent by p24 ELISA (Phillips D M et al., 1995, J Virol Methods, Mar,52 (1-2): 1-13). The results of this assay are provided below.

Cell-to-Cell Transmission (CTC) Assay

HIV-1-infected SupT1 cells (strain IIIB), killed by prior exposure tomitomycin C (200 μg/mL) for 1 hour at 37° C., were incubated with eachagent and P4-R5 (MAGI) cells for 2 hours at 37° C. Following theincubation period, cells were washed and provided with new media.Successful transmission and subsequent replication events werequantitated using Galacto-Star assay 48 hr post-infection. Infectedcells were expressed as a percent of cells infected after mock exposure.In each assay, triplicate wells were tested for each concentration. Theresults of this assay are provided herein.

Clinical Isolates in Peripheral Blood Mononuclear Cells (PBMCs) PBMCIsolation and Blasting

Peripheral blood mononuclear cells (PBMCs) were obtained from normalhepatitis and HIV-1 negative donors by ficoll hypaque gradientseparation. Briefly, anti-coagulated blood is diluted 1:1 withDulbecco's phosphate buffered saline without Ca⁺⁺ and Mg⁺⁺ (PBS) andlayered over 14 mL of Lymphocyte separation media in a 50 ml centrifugetube. Tubes were then centrifuged for 30 minutes at 600×g. Banded PBMCswere gently aspirated from the resulting interface and subsequentlywashed 2× with PBS by low speed centrifugation. The mononuclear cellswere counted, viability was determined by Trypan Blue dye exclusion andthe cells were resuspended in RPMI 1640 medium supplemented with 15% FBS(heat inactivated), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, and 10 μg/mL gentamycin with 20 U/mL recombinant IL-2 (R&DSystems, Minneapolis, Minn.). IL-2 was included in the culture medium tomaintain the cell division initiated by PHA mitogenic stimulation. Thecultures were then maintained until use by ½ culture volume change withfresh IL-2 containing medium every 3 days. Most assays were initiatedwith 3-day old blasted PBMCs. The results of this assay are providedherein.

PBMC Assay

Human peripheral blood mononuclear cells from a minimum of two donors,that have been stimulated with PHS- and IL-2, were counted and theirviability was determined by Trypan Blue dye exclusion and mixed in equalrations. Pooled donors were used to minimize the variability observedbetween individual donors, which results from quantitative andqualitative differences in HIV infection, and overall response to thePHA and IL-2 of primary lymphocyte populations. The cells wereresuspended at 1×10⁶ cells/mL in RPMI 1640 without phenol redsupplemented with 15% Fetal Bovine Serum (heat inactivated), 2 mML-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mLgentamycin and IL-2 (20 U/mL, R&D Systems, Minneapolis, Minn.). Fiftymicroliters of cells were then distributed to the inner 60 wells of a 96well round bottom microtiter culture plate in a standard format. Eachplate contained cell control wells (cells only), virus control wells(cells plus virus), and experimental wells (drug plus cells plus virus).Serially diluted compounds were added to the microtiter plate followedby the appropriate pre-titered strain of HIV-1. The plates were thenincubated for 6 hours at 37° C., 5% CO₂, centrifuged at approximately300×g for 10 minutes and 175 μL of supernatant removed with amulti-channel pipetor and replaced with fresh compound-free media. Thefinal volume per well was 200 μL. All samples were assayed in triplicatewith a replicate plate without virus for the determination of compoundtoxicity. The assay was incubated for 6 days in a humidified atmosphereat 37° C., 5% CO₂, after which supernatants were collected, for analysisof RT activity and sister plates analyzed for cell viability by XTT dyereduction. Wells were also examined microscopically and anyabnormalities were noted. The results of this assay are provided herein.

XTT Staining for Cell Viability and Compound Cytotoxicity

TC₅₀ values for the test materials were derived by measuring thereduction of the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) in replicate microtiter plates containing cell and compoundwithout virus. XTT in metabolically active cells was metabolized by themitochondrial enzyme NADPH oxidase to a soluble formazan product. XTTsolution was prepared daily as a stock of 1 mg/mL in PBS. Phenzainemethosulfate (PMS) solution was prepared at 15 mg/mL in PBS and storedin the dark at −20° C. XTT/PMS stock was prepared immediately before useof diluting the PMS 1:100 into PBS and adding 40 μL per mL of XTTsolution. Fifty microliters of XTT/PMS are added to each well of theplate and the plate incubated for 4 hour at 37° C. The 4 hour incubationwas empirically determined to be within the linear response range forMTS dye reduction with the indicated numbers of cells for each assay.Adhesive plate sealers were used in place of the lids, the sealed plateswere inverted several times to mix the soluble formazan product and theplates were read at 450 nm with a Molecular Devices SpectraMax Plus 96well plate format spectrophotometer. The results of this assay areprovided herein.

Reverse Transcriptase Assay

Reverse transcriptase activity, a measurement of virus replication, wasmeasured in cell-free supernatants. Tritiated thymidine triphosphate(NEN) (TFP) was resuspended in distilled H₂O at 5 Ci/mL. Poly rA andoligo dT were prepared as a stock solution which was kept at −20° C. TheRT reaction buffer was prepared fresh on a daily basis and consisted of125 μL 1.0 M EGTA, 125 μL dH₂O, 110 μL 10% SDS, 50 μL 1.0 M Tris (pH7.4), 50 μL 1.0 M DTT, and 40 μL 1.0 M MgCl₂. These three solutions weremixed together in a ratio of 2 parts TTP, 1 part poly rA:oligo dT, and 1part reaction buffer. Ten microliters (10 μL) of this reaction mixturewas placed in a round bottom microtiter plate and 15 μL of viruscontaining supernatant added and mixed. The plate was incubated at 37°C. for 60 minutes. Following reaction, the reaction volume was spottedonto pieces of DE81 paper, washed 5 times for 5 minutes each in a 5%sodium phosphate buffer, 2 times for 1 minute each in distilled water, 2times for 1 minute each in 70% ethanol, and then dried. Opti-Fluor 0 wasadded to each sample and incorporated radioactivity quantitatedutilizing a Wallac 1450 Microbetaplus liquid scintillation counter. Theresults of this assay are provided herein.

Infection of T Cells by HIV Associated to Dendritic Cells DC/T Assay

Monocyte-derived dendritic cells (MO-DC), infected with the NSI/R5strain Ba-L at 10-3 multiplicity of infection, were cultured alone orwith autologous CD4(+) T cells at a ratio 1:1 or 1:10. A dilution seriesof suramin was added to the MO-DC, 1 hour prior to infection andremained present during infection. After infection, cells wereextensively washed and compounds were added at the same concentrationsas for the pre-incubation. Medium and compounds were refreshed twice aweek. After 2 weeks, HIV Ag was measured in supernatants with ELISA andEC₅₀ values (effective conc. 50%) were calculated using linearregression (Vanham G et al, AIDS 2000, 14: 2299-2311). The results ofthis assay are provided herein.

Results

Suramin has previously been reported to confer protection to T cells invitro against the infectivity, replication and cytopathicity caused byhuman T-cell lympotropic virus (HTLV-M) (Mitsuya H et al. Science,October; 226(4671): 172-4, 1984; Balzarini J. et al. Int. J. Cancer.,37: 451-7, 1986). Furthermore, it was tested in patients with Kaposi'sSarcoma or AIDS-related complex with a partially successful outcome(Broder S. et al., Lancet., 2(5456):627-30, 1985). However, suramin wasnever evaluated or intended for use as an intravaginal or intrarectalmethod to prevent sexual transmission of HIV. Systemic toxicity and apoor therapeutic index were the main reasons not to continue withclinical trials. These reasons, as well as suramin's long half life andhigh protein binding, precluded others from identifying a topicalapplication of suramin to prevent sexual transmission.

Suramin sodium was found herein to be highly active against lympho- andmonocytotropic strains of HIV-1 (IC₅₀=7.3 μg/mL [72 hours incubation]and 18.5 μg/mL [2 h-incubation] against IIIB in viral entry assays and0.34 and 5.5 μg/mL against Bal [2h-incubation]) (FIG. 5). It was alsoeffective in a CD4-independent epithelial cell transmission assay(ME-180 assay; IC₅₀=24.5 μg/ml.) (FIG. 6), a CD4-dependent cell-to-celltransmission assay (CTC assay; IC₅₀=93.5 μg/mL) (FIG. 5) and in thedendritic cell (DC)/T lymphocyte model (IC₅₀=17 μg/mL) (results shown inTable 7).

TABLE 7 Inhibition of HIV Transmission from Dendritic Cells to T CellsCells Effecting Transmission Dendritic DC + T Cells DC + T CellsCompound Cells (DC) (1:1) (1:10) Suramin 56 ± 6 57.5 ± 5 17 ± 3 (EC₅₀ inμg/mL) DC infected with BaL (R5 HIV-1) were cultured alone or incombination with autologous T lympocytes at two ratios in the presenceof multiple concentrations of suramin.

Addition of mucin in the ME-180 assay to resemble cervicovaginalsecretions did not significantly reduce suramin's activity (FIG. 6).Formulating suramin at 2% in KY Jelly® maintained its antiviral activityintact. Interestingly, formulating suramin in Replens®, a carbopol-basedvaginal lubricant, had a significant synergistic effect, especially insuramin's antiviral activity against cell-free and cell-associated HIV-1IIIB (Table 8).

TABLE 8 Anti-HIV Activity of Suramin Formulations Cell-Free StrainsCell-Associated Strains IIIB BaL IIIB Compound (mg/mL) (mg/mL) (mg/mL)Suramin 18.5 5.5 93.5 2% suramin in KY 24.4 3.16 287.5 Jelly ® 2%suramin in 2.1 7.2 9.6 Replens ® Positive control 1.6 14.6 12.9 Datarepresent 50% inhibitory concentrations (IC₅₀) Target cells areCD4/coreceptor expressing HeLa cells. Incubation time: 2 hours

The preventative aspects of the compositions and methods of theinvention have not been fully appreciated prior to the instantinvention. For example, prior to the present invention, it was knownthat HIV infects the vaginal mucosa in less than 2 hours after contact(Hu J et al., J Virol 2000 July; 74(13): 6087-95), and the rectal mucosalikely in a shorter time. However, all the reported assays purporting todescribe suramin anti-HIV activity used prolonged incubations (days),wherein the compound was present during the entire incubation period.While appropriate for a therapeutic application, this mode of evaluateis inappropriate for a method of describing the prevention of vaginal orrectal transmission of HIV, where the agent should act during a windowof infectivity that starts at the time of sexual contact and ends assoon as the compound falls below effective concentrations (e.g., due toleakage and dilution) or the virus is inactivated by the renewed acidicvaginal pH.

Moreover, epithelial cells and dendritic cells, present in vaginal andrectal mucosa, are capable of being infected or transmit live virus evenafter several days after the initial infection. It has now beendiscovered that suramin is capable of blocking CD4-independentepithelial and dendritic cell infection, two events without whichprevention of sexual transmission would not be possible.

In order to prevent mucosal transmission of HIV, includingcervical-vaginal HIV infections as described herein, a variety of keyexperimental data regarding the function and effectiveness of thecompositions should be obtained. For example, the compound orcomposition should a) inactivate the virus or inhibit HIV entry withinless than 2 hours of contact with the virus, for that is the acceptedtime frame in which the virus infects the vaginal mucosa; b) beeffective against cell-associated virus, which is a critical componentof the HIV load in semen (i.e., block cell-to-cell transmission); c)block infection of epithelial cells, as well as CD-4-bearing cells; d)block HIV infection of and transmission by dendritic cells (one of theprimary targets in mucosal transmission); and e) inhibit monocytotropicHIV-1 strains (predominant in sexual transmission).

It has been demonstrated herein that suramin possesses all theabove-mentioned characteristics. Furthermore, it has also been shownherein that suramin is not toxic to the vaginal epithelium orlactobacilli, hydrogen peroxide producing bacteria that control thegrowth of pathogenic microorganisms, and displays anti-inflammatoryactivity on vaginal cells (i.e., inhibits vaginal secretion ofproinflammatory cytokines). This latter finding makes suramin an idealagent to combine with surfactants, virus inactivators, antimicrobials,and microbicides which, because of their nature, display proinflammatoryactivity, a feature that favors HIV mucosal transmission. Moreover, ithas not been demonstrated that suramin, when administered vaginally,exhibits negligible bioavailablity.

If a compound does not have the above-mentioned characteristics, oneskilled in the art would recognize that the compound or composition cannot be recommended as a prophylactic for sexual transmission of HIV.

Activity Against HSV

Evaluation of anti-HSV activity of suramin was based on the methoddescribed by Herold et al., J. Virol., 70: 3461-3469 (1996). Thecompound was serially diluted (1000 μg/ml to 1.0 μg/ml) inphosphate-buffered saline (PBS), and each dilution was mixed with eachof two serial dilutions (10⁻⁵ and 10⁻⁶) HSV (type 2 strain 333). Workingviral titers were approximately 10³ and 10⁴ plaque forming units (pfu)per ml, respectively. Samples (1 ml) of each mixture were plated induplicate on washed and drained confluent monolayers of CaSki cells (ahuman cervical epithelial cell line, obtained from the American TypeCulture Collection, Rockville, Md.) on the bottoms of 25 cm² flasks.Initial titer (indicated in the control cultures with no added testagent) in each culture was given as pfu/ml. The flasks were incubatedfor 2 hours, after which the medium (containing virus and test compound)was removed and the cells were washed with PBS. Cells were cultured for3 days in medium 199, supplemented with 1% serum and 0.5% methylcellulose that contained anti-HSV antibodies to prevent the formation ofsecondary plaques. Cells were stained with Giemsa for the counting ofviral plaques by phase contrast microscopy. The viral titer was inferredfrom the number of plaques, after correction for dilutions. Values werecorrected to a control titer 5×10⁸ pfu/ml. Data were expressed as viraltiter and as the percentage of plaques that are counted in control cellcultures (not exposed to test agent). Test compounds that showed noactivity at 100 μg/ml were considered inactive as anti-HSV agents. Theconcentration of the compound that was required to reduce the viraltiter by 50% (IC₅₀) was estimated with curve-fitting software(TableCurve 2D, version 3.02) from plots of plaque-forming units (pfu)per ml as a function of concentration of the compound.

Suramin sodium was highly effective against HSV-2 as well, exhibiting100% inhibition of viral infectivity at 100 μg/mL in a plaque assay.Results are shown in FIG. 7. While not being significantly cytotoxic atthe highest concentration tested (1000 μg/mL), suramin's IC₅₀ againstHSV-2 and HSV-1 were 1 and 30 μg/mL, respectively.

Activity Against Cytomegalovirus

In a cytomegalovirus binding assay, tritiated murine cytomegalovirus(MCMV) was incubated with NIH3T3 fibroblasts in a 24-well plate fixed(0.4% paraformaldehyde) for 2 hours at 37° C. (rocking) in the presenceof media alone or media containing 1-1000 μg/mL suramin. Cells were thenwashed extensively and lysed with 1% SDS and triton X-100. Lysates werequantified for radioactivity (cpm) in a scintillation counter.

Suramin sodium was also effective against CMV, displaying an IC₅₀=50μg/mL in a binding assay, as seen in FIG. 8.

Activity Against Chlamydia trachomatis (CT)

Chlamydia multiplication inhibition was performed according to Cooper M.D. et al., J. Gen. Microbiol., 136: 1109-1115 (1990). Prior toexperimentation, cryo-preserved Chlamydia trachomatis (serotypeE/UW-5/CX) were quickly thawed (37° C.) and suspended by mildsonication. Serial 1:10 dilutions of the bacterial suspension were made,ranging from 10⁻¹ to 10⁻⁷.

HeLa monolayers (on coverslips) were inoculated with 100 μl of thedifferent dilutions of chlamydia (elementary bodies), in the presence orabsence of test agent. The suramin compound was tested at 0.1, 1.0, 10,100 and 1000 μg/ml. After 1 hour, the monolayers were washed to removefree chlamydia and test agent, and they were incubated for 48 hours.

Medium was removed, and the HeLa monolayers were fixed in methanol,washed, and treated with Kallsted Chlamydia Culture Confirmationfluorescein-conjugated Monoclonal Antibody. Reaction of labeled antibodywith the cells was carried out for 30 minutes at room temperature (i.e.,25° C.) (no light) in a humidified chamber. The cells were washed withwater; a drop of mounting medium is placed on a glass slide, and thecoverslip was applied. Inclusions due to chlamydial infection werevisualized with a fluorescent microscope as green fluorescence. Data wasreported as the number of inclusion forming units per ml of undilutedchlamydial titer, and were adjusted to control values of 2.6×10⁷ IFU/mlfor control cultures (no test agent) in each experiment. Compounds thatshow no activity at 1000 μg/ml are considered to be inactive asanti-chlamydial agents. After logarithmic transformation of all data,dose-response curves were created with curve-fitting (TableCurve 2D,version 3.02; Jandel Scientific) software.

Suramin sodium inhibited CT multiplication by 75% at 100 μg/mL (IC₅₀=28μl/mL) (FIG. 9). At 2 mg/mL, inhibition was essentially complete(control titer: 1×10⁻⁵, suramin sodium titer: 0.08×10⁻⁵).

Neisseria gonorrhoeae (GC)

Gonococcal Growth Inhibition

Gonococcal growth inhibition assays were performed by the method ofAnderson R. A., et al., J. Androl., 21: 862-875; 2000. Briefly,Neisseria gonorrhea from local uncomplicated cases of gonorrhea wereisolated (verified by Gram stain, oxidase reactivity and sugarfermentation). The titer of log-phase cultures were adjusted by dilutionin GC broth to 0.5 McFarland standard (approximately 10⁸ colony formingunits per ml). This was diluted 1:10 with broth that contained noadditions (control), and each of serial 1:10 dilutions of test agent,ranging from 1 mg/ml to 1.0 μg/ml. The cultures were incubated at 37° C.for four hours. Four serial 1:10 dilutions were made in GC broth, and aportion of each dilution was inoculated onto GC agar plates. The plateswere incubated overnight, and resultant gonococcal colonies were countedunder bright-field microscopy. Data for each concentration of suraminwere expressed as the number of colony-forming units (CFU) per ml of theoriginal log-phase bacterial suspension.

Referring to FIG. 10 (testing of suramin tested at 1-1000 μg/mL), therewas no clear dose-response. However, suramin sodium completely inhibitedGC growth (multiplication assay) at 1 mg/mL.

A summary of the antimicrobial activity of the suramin compositions ofthe present invention is provided in Table 9.

TABLE 9 Summary of antimicrobial activity of suramin compositions. Typeof Inhibition/ Activity Inactivation Assays Potency CommentsAnti-Microbial HIV-1 VBIA-IIIb High IC₅₀ = 7.3 μg/mL (in vitro) MAGI-BalHigh IC₅₀ = 0.39 μg/mL ME-180 High IC₅₀ = 24.5 μg/mL Clin Isolates HighIC₅₀ = 14.6 μg/mL CTC High IC₅₀ = 93.5 μg/mL (API) and 9.6 μg/mL DC HighIC₅₀ = 17 μg/mL SIV Reverse High IC₅₀ = 4 μg/mL transcriptase HSV-2Plaque High IC₅₀ = 1 μg/mL (HSV-2) and 30 μg/mL (HSV-1) CMV BindingModerate IC₅₀ = 50 μg/mL Chlamydia Multiplication High IC₅₀ = 28 μg/mL;100% (CT) inhibition at 2 mg/mL Gonococcus Multiplication Moderate 100%inhibition at 1 mg/mL. No (GC) dose-response

It is believed that this is the first report of suramin displayingantibacterial activity. Suramin showed inhibiting activity againstChlamydia trachomatis and Neisseria gonorrhoeae, two of the mostprevalent sexually transmitted pathogens. This antibacterial activity ofsuramin could not have been predicted based on its known antiviral ortrypanocidal activity.

Example 3 Local Toxicity Vaginal Cytotoxicity

Vaginal cytotoxicity was determined by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)assay and cytokine assays, both as described in Fichorova R. N. et al.,J. Infect. Dis. 184(4): 418-28, 2001). The MTT assay assesses thecytotoxicity of various compounds against cell lines by plating thedesired cells prior to exposure to the compounds. On the day oftreatment, culture medium was carefully aspirated from the wells andreplaced with fresh medium containing the test compound at variousconcentrations. The cells were incubated with the various compounds for6 hours, washed with fresh medium and MTT was then added and the plateswere incubated at 37° C. for a period of time sufficient to allow MTT toform formazan crystals by reacting with metabolically active cells. Theformazan crystals were solubilized overnight at 37° C. in a solutioncontaining 10% SDS in 0.01 M HCl. The absorbance of each well wasmeasured in a microplate reader (Dynex) at 540 nm and a referencewavelength of 690 nm.

Interleukin levels were assessed in the supernatant collected after 6hours of compound-cells incubation using commercial ELISA kits (R&DDiagnostics, Minneapolis, Minn.) according to manufacturer'sinstructions (also see Fichorova R. N. et al., J. Infect. Dis. 184(4):418-28, 2001).

Incubated with a human vaginal cell line (VK2) for 30 minutes, 6 hours,and 24 hours at concentrations ranging from 10 mg/mL to 10 μg/mL in aMTT assay, as described above, suramin sodium showed no evidence ofcytotoxicity (FIG. 11). This finding was reinforced by the lack ofcytotoxic effects described in the antimicrobial assays using differentcell lines such as MT-2, HeLa, CaSKi, and others. In a comparative studywith dextran sulfate, cellulose sulfate, and N-9, suramin sodium was theleast cytotoxic of them all. Another element that enables the use ofsuramin formulations as a vaginal method to prevent sexual transmissionof HIV is the demonstration of its lack of deleterious effects on thevaginal environment. Suramin was proved to be safe to the vaginalepithelial lining and the lactobacilli (beneficial bacterial that keeppathogenic bacterial growth in check). Furthermore, suramin did notinduce a proinflammatory reaction, and reduced the inflammation causedby surfactants. Given that surfactants like nonoxynol-9 have failed inclinical trials, not because of their poor virucidal activity but,because of the inflammation they caused (Van Damme L. et al., Lancet.,360(9338): 971-7, 2002; Fichorova R. N. et al., J. Infect. Dis. 184(4):418-28, 2001), a combination with suramin, which displayedanti-inflammatory activity, appears to be ideal.

Proinflammatory Cytokines

Suramin sodium did not induce the release of IL-1β by cultured vaginalcells in an ELISA assay. This was in sharp contrast with N-9's and othersurfactants' effects. Similar results were obtained with IL-1a IL-6,IL-8, and IL-18 (data not shown) as partially seen in FIG. 12. There wasno stimulation of proinflammatory cytokines. Furthermore, suramin sodiumdecreased N-9-induced release of IL-1 and IL-8 by vaginal cells,therefore exerting anti-inflammatory activity (FIG. 12). This activityshould be of relevance to decrease the number of leukocytes that serveas targets of HIV infection.

Lactobacilli Growth

Suramin sodium evaluated at 5 mg/mL (highest concentration tested) hadno inhibitory effect on the growth of Lactobacillus gasseri (growth was107% of control's) (FIG. 13).

A summary of the local toxicity activity is presented in the followingTable 10:

TABLE 10 Local Toxicity Activity of Suramin Compositions. Type ofInhibition/ Activity Inactivation Assays Potency Comments Local ToxicityLactobacilli Doubling time None Max. concentration tested = 5 (in vitro)mg/mL Vaginal Cell MTT None Max. concentration tested = 10 Cytotoxicitymg/mL (various cell types) Proinflammatory ELISA None IL-1β, IL-1∀,IL-8, Cytokines IL-6, IL-18 (decreases N-9 induced cytokine release)

Example 4 Pharmacokinetics

Given that suramin had been abandoned as a therapeutic agent due ingreat part to its systemic adverse-effects, it was critical to thefeasibility of its use as a vaginal preventative anti-STD pathogenmethod (this application) to demonstrate low bioavailability from thevaginal compartment.

Female rats (n=6) were administered with 30 mg of compound per kg in asingle I.V. dose in saline or intravaginally, BID, for 5 days,formulated in KY jelly. The selected single dose represented 10 timesthe estimated human dose (250 mg). No clinical signs were observedduring the conduct of this study. Following intravenous administration,relatively high concentrations of suramin sodium (mean value=563 μg/mL)were measured in plasma at 1 hour (first time-point) following the onsetof dosing. Suramin sodium appeared to be eliminated slowly followingthis route of administration as suggested by the long mean half-lifevalue (68.5 hr) and the small mean clearance value (1.75 mL/hr). Themean volume of distribution was calculated at 147 mL/kg.

Following intravaginal administration, relatively low concentrations ofthe compound (mean value=17.7 μg/mL) were measured in plasma at 3.5hours following the last dose. Suramin sodium appeared to be eliminatedslowly following intravaginal administration as suggested by the longmean half-life value (132 hr, 5.5 days) and the small mean clearancevalue (0.006 mL/hr).

The bioavailability calculated from the total dose (90 mg) administeredintravaginally was negligible at 0.8%. No histological lesions wereobserved in selected tissues (vagina, lungs, liver and kidneys) offemale rats receiving the intravenous solution of the compound.

Due to very poor systemic absorption after oral administration, suraminhas been administered intravenously in the past. Furthermore, due to itshigh protein-binding affinity, it was administered in large (gram)quantities. Large doses of suramin I.V. showed side-effects whichreduced its therapeutic index as a systemic anti-HIV drug. Thus,clinical trials were not expanded, and the compound was not pursuedfurther. However, in the proposed use as a mucosal (vaginal) microbicideand contraceptive, poor systemic absorption is an advantage, since itlimits its action to the site of administration and prevents thedevelopment of systemic side-effects. As described above,pharmacokinetics studies performed demonstrated negligible (<1%)bioavailability after vaginal administration.

A summary of the pharmacokinetics analysis is provided in Table 11.

TABLE 11 Summary of Pharmacokinetics Analysis, showing the mean andindividual pharmacokinetic parameters of suramin in plasma of femalerats following intravenous infusion and intravaginal administration at adose of 30 mg/kg Cmax AUC(tf) AUC(I) t½ Vdss CL Group Kel (μg/mL)(μg-hr/mL) (μg-hr/mL) (hr) (mL/kg) (mL/hr) IV 0.0102 563 13857 1728968.6 147 1.75 Ivag 0.005 17.7 1174 2275 2275 132 0.006 IV: A singleintravenous dose of 30 mg/kg IVag: Intravaginal doses of 30 mg/kg BIDfor 5 days AUC(I): The area under the plasma concentration vs time curvefrom time zero to infinity AUC(tf): The area under the plasmaconcentration vs time curve from time zero to 144 hr post dose CL:Plasma clearance Cmax: The highest observable concentration Kel:Elimination constent Tmax: Time to Cmax t½ Terminal phase half-lifeVdss: Apparent volume of distribution at steady state

All publications mentioned in this specification are herein incorporatedby reference, to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1.-35. (canceled)
 36. A topical composition, comprising a pharmaceutically acceptable carrier and an amount of a surfactant and either suramin or a pharmaceutical salt thereof effective to inhibit transmission of sexually transmitted diseases and inhibit sperm-egg fertilization.
 37. The composition of claim 36, wherein said surfactant is selected from the group consisting of nonionic surfactants, cationic surfactants and anionic surfactants.
 38. The composition of claim 37, wherein said surfactant is selected from the group consisting of sorbitan monolaurate, nonylphenoxypolyethoxy ethanol, p-diisobutyphenoxypolyethoxy ethanol, polyoxyethylene (10) oleyl ether, onyx-ol, sodium alkyl sulfonates, sodium alkylbenzene sulfonates, alkyl ketones, cetyl pyrimidinium chloride, and benzalkonium chlorides. 39.-40. (canceled)
 41. A method for simultaneously inhibiting sexually transmitted infections and inhibiting sperm-egg fertilization comprising administering to a female intravaginally, to the cervix or to the uterus a composition comprising suramin in an amount effective to inhibit sexually transmitted infections and sperm-egg fertilization, wherein said sexually transmitted infections are caused by a microorganism selected from the group consisting of Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Calymmatobacterium granulomatis, Mycoplasma gentitalium, and Ureaplasma urealyticum.
 42. The method of claim 41, wherein said microorganism is selected from the group consisting of Neisseria gonorrhoeae and Chlamydia trachomatis. 43.-50. (canceled)
 51. The composition of claim 38, wherein said surfactant is sorbitan monolaurate.
 52. The composition of claim 36, wherein said sexually transmitted diseases are caused by a bacteria selected from the group consisting of Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Calymmatobacterium granulomatis, Mycoplasma gentitalium, and Ureaplasma urealyticum.
 53. The composition of claim 36, wherein said composition exerts anti-inflammatory activity.
 54. The composition of claim 36, wherein said composition inhibits vaginal secretion of proinflammatory cytokines.
 55. The composition of claim 36, wherein a single administration corresponds to a total dose of about 5 to 100 mg/gram.
 56. A device for administering the composition of claim 36 to the vagina or uterus, comprising a solid support adapted to be inserted into the vagina, said support being impregnated with or coated with a composition comprising either suramin or a pharmaceutical salt thereof and a pharmaceutically acceptable carrier.
 57. A topical composition, comprising a pharmaceutically acceptable carrier and an amount of either suramin or a pharmaceutical salt thereof effective to inhibit transmission of sexually transmitted diseases and inhibit sperm-egg fertilization.
 58. The composition of claim 57, further comprising a surfactant selected from the group consisting of nonionic surfactants, cationic surfactants and anionic surfactants.
 59. The composition of claim 58, wherein said surfactant is selected from the group consisting of sorbitan monolaurate, nonylphenoxypolyethoxy ethanol, p-diisobutyphenoxypolyethoxy ethanol, polyoxyethylene (10) oleyl ether, onyx-ol, sodium alkyl sulfonates, sodium alkylbenzene sulfonates, alkyl ketones, cetyl pyrimidinium chloride, and benzalkonium chlorides.
 60. The composition of claim 57, wherein said composition exerts anti-inflammatory activity.
 61. The composition of claim 57, wherein said composition inhibits vaginal secretion of proinflammatory cytokines.
 62. The composition of claim 57, wherein a single administration corresponds to a total dose of about 5 to 100 mg/gram.
 63. A device for administering the composition of claim 57 to the vagina or uterus in a form selected from the group consisting of liquid, lotion, oil-in-water emulsion, water-in-oil emulsion, liposomes, gel, foam, film, spray, ointment, pessary, suppository, capsule, tablet, jelly, and cream.
 64. A device for administering the composition of claim 57 to the vagina or uterus, comprising a solid support adapted to be inserted into the vagina, said support being impregnated with or coated with a composition comprising either suramin or a pharmaceutical salt thereof and a pharmaceutically acceptable carrier.
 65. The device of claim 64, wherein said solid support is selected from the group consisting of a sponge, a cervical cap, a diaphragm and an intrauterine device. 